Technology for the analysis of clinical specimens has provided significant advances in the field of medicine and public health. For example, it is now possible to routinely diagnose many clinical conditions using a wide variety of assays which determine qualitative and/or quantitative characteristics of a specimen. The methods of detecting multiple viral infections of the same viral group through a single assay have thus far shown only very limited capability and questionable results. A variety of reasons have been mentioned for these limitations, including the particularly difficult solution of standardizing an immunoassay for heterogeneous antigens. Additional reasons include the extended times typically required to enable the detection and classification of multiple viral infections of the same viral group, unwieldy collection, classification and analysis in the algorithms analyzing the data, and the inability to positively identify more than one viral infection in the same test. Previous multiple viral detection methods have been able to identify one viral infection and distinguish secondary viral infections by a process of elimination. A single assay able to efficiently and accurately positively detect a single viral infection from two candidate viral infections of the same viral group in a clinical sample would clearly be an improvement in the field. The capability to perform simultaneous, multiple determinations in a single assay process is known as “multiplexing” and the method of utilizing such determinations is “multiplexing analysis.”
Microsphere “bead”-based (microparticle) immunoassays (MIAs) are becoming increasingly popular as a serological option for laboratory diagnosis of many diseases. The technology involves the detection and analysis of a reaction attached to microparticles. The common detecting instrument is a simplified flow cytometer, which has lasers that simultaneously identify the microparticle sets and measure the fluorescence associated with the reaction. Previous attempts have used microparticles coupled to recombinant envelope and nonstructural proteins, but such attempts cannot concurrently positively diagnose multiple viral infections of the same viral group in a single assay.
The more traditional serological method for identifying an infecting virus is the time-consuming and technically difficult plaque-reduction neutralization test (PRNT). The serological testing algorithm in common usage in the U.S. state health departments uses the immunoglobulin M (IgM) antibody capture enzyme-linked immunosorbent assay (MAC-ELISA) and the indirect immunoglobulin G (IgG) ELISA as primary tests following by confirmatory PRNT tests for positive samples from the ELISA testing. IgG antibodies to viruses within the same serocomplex exhibit extensive cross-reactivity, whereas IgM antibodies are less cross-reactive. The MAC-ELISA is a 2-day test that requires 4 hours of hands-on time for a 40-sample test. This combination of assays is highly sensitive and specific, but can require in total 2-3 days to complete, as overnight incubations are deemed necessary to enhance sensitivity. Thus, the advent of a more rapid, yet equally sensitive, single test to replace separate ELISA tests to detect a single viral infection from two candidate viral infections of the same viral group in a clinical sample would be a great benefit to addressing public health needs. Many viruses can be transmitted through blood transfusion and organ transplantation, further heightening the urgency and need for the development of specific and rapid serological assays of a single viral infection considered from viruses of the same viral group.